Effects of mulberry leaf enrichment with Lepidium sativum L. seed powder suspension on the economic parameters of Bombyx mori L

The phytochemicals of high nutritional and functional properties in Lepidium sativum L. (garden cress) seeds have nominated their seed powder (regardless of the concentration used) for enrichment of mulberry leaves in order to enhance Bombyx mori L. larval feeding, and consequently to gain ground in sericulture industry. As expected, B. mori larval feeding on L. sativum-enriched mulberry leaves showed not only a remarkable increase in mean values of certain economic parameters of B. mori, such as cocoon weight, cocoon shell weight, pupal weight, and egg yield, compared with the control group, but also showed a phenomenal increase in egg counts (on average, ca. 958–1256 eggs laid per female moth) and a significant increase in egg size (measured as egg surface area and egg volume). Male or female moth larval diet has significantly influenced the reproductive performance or fitness of both sexes of B. mori in terms of large-sized moths (measured as forewing, hind femur, and hind tibia lengths) and highly fecund moths (i.e., increased fecundity and spermatophore counts per female moth, and large-sized eggs). On the basis of B. mori female moth reproductive index, the female moths from L. sativum-fed larvae proved to have a lower reproductive index compared to their corresponding value for females of the control group, indicating more efficient utilization of larval resources for B. mori reproduction. Quantification of the three main physiological resources viz., protein, lipid and carbohydrate in the internal reproductive tract of B. mori female moths at death has nominated the female moth abdomens, or simply their bodies, as being a reasonable natural source of protein, lipid, and carbohydrate, to be involved in certain manufactures (e.g., pet feed formulations) instead of discarding them as a source of environmental pollution. Evidently, the L. sativum seed powder is of considerable interest because it remarkably improves the performance of such an economically important insect, B. mori. This is the first study for evaluating the efficacy of L. sativum seed powder in sericulture field to enhance B. mori productivity parameters.

L. sativum seeds used in this study were obtained from the local market of the type grown in Al-Qaseem area in Saudi Arabia, and authenticated by an expert taxonomist.The seeds were ground by an electric grinder to get a fine powder which was stored in an airtight glass jar.Three concentrations (0.25, 0.50, and 1.00%, w/v) were prepared in distilled water.Based on the tested concentration, the calibrated weight of L. sativum seed powder and distilled water were thoroughly dissolved in a suitable container until getting a sort of a homogenous suspension.Fresh and clean mulberry leaves were dipped, separately, in the tested concentration for ca. 1 min (with stirring in order to ensure a complete coverage of the tested suspension for the leaf-upper and lower-surface) and air-dried for 5-10 min.
B. mori larvae were allocated into three groups; the first group served as the control, the second and the third groups served as the treatments.Control larvae were fed (from egg hatching till cocoon spinning) on mulberry leaves treated with distilled water only.Treatment-larvae of the second group were fed (from egg hatching till cocoon spinning) continuously on mulberry leaves enriched with L. sativum seed powder suspension of each tested concentration (continuous feeding); whereas B. mori larvae of the third group were fed on treated leaves for 48-h interval, then untreated mulberry leaves were supplied for 48-h interval; then treated mulberry leaves were supplied for another 48-h interval, and so on till cocoon spinning (discontinuous feeding).All the treatments and the control were replicated thrice, and each replication consisted of 100 larvae.
The observations were recorded on the following economic and physiological parameters: cocoon weight, cocoon shell weight, cocoon shell ratio percentage, pupal weight, adult longevity, female moth fecundity (number of deposited eggs per female), number of spermatophores per female moth, and egg size (i.e., egg length and width; egg surface area and volume).Egg volume was estimated as a prolate spheroid, using the formula V = 4/3 π abc (a;b;c are the radii at different planes 8 ).Dimensions of the egg (a, b, c) were measured using an ocular micrometer on a dissecting binocular microscope (20X).The egg surface area was calculated using the formula of an ellipse (A = π ab; where a = length of semimajor axis and b = length of semiminor axis 9 ).Also, the effect of body size of male and female moths, and number of matings (number of spermatophores per female) on female moth reproductive output (fecundity and longevity) were evaluated.The lengths of forewing, hind femur, or hind tibia (as indices of body size) were measured using both of a millimeter paper for scale and a dissecting binocular microscope at 20X 10,11 .The total protein-, lipid-and carbohydrate-content of female moth internal reproductive tract (ca.60 females per treatment) was also quantified directly after their death.The total protein, lipid, and carbohydrate contents were estimated from frozen samples (− 20 °C) of the female moth internal reproductive organ homogenates by using the Kjeldahl method and Soxhlet 12,13 .

Statistical analysis
Results were statistically analyzed using the ANOVA followed by Duncan's multiple range test at 5% probability level.The results were represented as Mean ± SE.Statistical analyses were performed using the IBM SPSS Statistics for Windows, Version 20, Armank, NY, IBM Crop, 2011.

Performance of B. mori in the presence of L. sativum seed powder in its larval diet
The effects of enrichment of mulberry leaves with L. sativum seed powder on B. mori performance (pupal and adult parameters) are summarized in Tables 1, 2, 3, 4, 5, 6 and 7. www.nature.com/scientificreports/

Pupal economic parameters
As shown in Tables 1 and 4, the recorded significant increases in cocoon parameters, cocoon weight, pupal weight, and cocoon shell weight, at all indicated treatments were remarkably better than the control groups.The percent increase values over their control values ranged approximately between 24.4-38.5%,34.2-51.0%,and 5.9-14.4%,respectively.Only a marginal decrease (− 0.34%), but statistically not significant, was recorded for the cocoon shell weight obtained from cocoons produced by B. mori larvae fed, discontinuously, on mulberry leaves enriched with a 1% L. sativum (0.320 ± 0.005 gm) when compared to the control group (0.321 ± 0.005 gm) (Table 1).On the other hand, the cocoon shell ratio (%) in the control group was significantly higher than that of the L. sativum-fed group.The reduction ranged, nearly, from 15.3% to 24.1% (Table 1).This reduction could be attributed to the positive increase of pupal weights in the experimental groups (Tables 1, 4) as compared to the significantly low pupal weights in the control group.In general, no obvious trend was observed in this study to address the effect of both the L. sativum-concentration used and the experimental larval feeding pattern (i.e., continuous or discontinuous) on the studied cocoon parameters.Meanwhile, higher mean values of both the cocoon weight and the pupal weight were evident when B. mori larvae were fed continuously on mulberry leaves enriched with L. sativum; irrespective of L. sativum-concentration used (Tables 1, 4).

Fecundity (no. of eggs laid per female)
As shown in Tables 2 and 5, B. mori larval feeding, continuously or discontinuously, on L. sativum-enriched mulberry leaves leads to a significant increases in fecundity by nearly two to threefold or 62.26-202.87% in the experimental groups when compared to the control group.Evidently, the maximum increases of 177.42-202.87% in fecundity or the egg yield averages (1250.14 ± 78.65-1364.79± 43.46 egg) were accompanied with L. sativumconcentration higher than 0.25% (i.e., 0.50 or 1.00%).It appears, to a large extent that irrespective of the L. sativum-tested concentration, low or high, the highest egg yield averages were recorded in B. mori-discontinuous larval feeding pattern of the experimental group (Tables 2, 5).

Egg size
As an index of egg size, the egg length, width, volume, and surface area were estimated.B. mori larval feeding on L. sativum-enriched mulberry leaves considerably affected the estimated egg parameters.In the experimental group, mean values of these parameters were significantly increased when compared to the control group (Tables 3, 5).On calculating the percent increase values over their control ones, a minimal increase of 6.5% and a maximal one of 9.5% were recorded in the egg length; whereas the corresponding values for the egg width were 11.4% and 17.8% (Table 5).Also, the egg surface area and volume were increased, ca., by 33.65-61.5% and 18.6-31.1%,respectively (Tables 3, 5).Such an obvious percent increase in the egg size parameters over the control was, in general, a conc.-dependent and a larval feeding pattern-dependent as well.In other words, www.nature.com/scientificreports/higher increases were always associated with both of higher concentrations of L. sativum seed powder and the larval continuous feeding pattern.

Male or female moth body size
The lengths of forewing, hind femur, and hind tibia were measured as an index of B. mori male or female moth body size.As shown in Tables 3 and 5, the average body size of the male or the female, was significantly increased.The forewing mean lengths were increased by up to 5.07-11.88%or 5.67-28.11% in the male or the female moths, in respect (Table 3).Among the calculated mean values of female or male hind femur length, the percent increase, in the treatments, over the control had ranged from 2.06 to 13.27% in female moths, or ranged from 2.15 to 7.60% in male moths (Table 3).Also, when the female or male moth body size was measured as a hind tibia length, approximately similar increases in the mean values of hind tibia lengths occurred by up to 2.76-15.46% in the female, or 4.92-8.22% in the male (Table 3).As is evident from the data presented in Tables 3 and 5, the body size indices of both sexes were, in general, significantly increased in length in a conc.-dependentway, and a larval feeding patterndependent as well; where, in general, it appears that the observed increase in the above-mentioned indices was associated with B. mori-discontinuous larval feeding on L. sativum-enriched mulberry leaves.However, few exceptions did not follow such a dependency (Tables 3, 5).

Longevity of female and male moths
In the experimental group, the female moth longevity recorded an increase of 2.22-9.81%;however, this increase was not significantly different to that of the control group.Also, an increase of 9.73-39.40%was recorded for the male moth longevity, in the treatments; but such increases were significantly different to that of the control except www.nature.com/scientificreports/for the male longevity at conc.0.50% (Table 3).In terms of male or female moth longevity, such increases, significant or insignificant, seemed to be neither a dose-dependent nor a larval feeding pattern-dependent (Table 3).

Relationship between spermatophore counts and egg counts
In terms of spermatophore counts in the bursa copulatrix of B. mori-mated females, the number of matings by their males ranged from one to two (mean value = 1.37) in the control group; whereas the corresponding number of matings in the experimental group ranged from one to six (mean values = 2.26-3.00),but never mate more than six times.The differences in mean spermatophore numbers between the two groups, the control and the experimental, were significant in favour of the latter group (Table 2).
Based on the calculated values of the percent increase, in the experimental group, over that of the control one, the mean spermatophore counts per female showed to be increased by up to 64.54-118.42%(Table 2).Also, the results show that in the control group, 62.65% or 37.35% of B. mori males were able to mate once or twice, respectively.In the experimental group, at conc.0.25% and a continuous larval feeding pattern, 3.90, 24.68, 24.68, 29.87, and 10.39% of males could achieve one, two, three, four, and five matings, respectively; whereas with the discontinuous larval feeding pattern, the corresponding percentages of one, two, three, four, and five matings were, in respect, 1.67, 31.67,38.33, 21.67, and 6.67%.At the tested conc. of 0.50 or 1.00%, whether at the continuous or the discontinuous larval feeding pattern, the number of matings for males ranged from one to four.Only one male (1.45%) was able to achieve six matings.The maximum mean number of spermatophores per female (ca., 3 spermatophores) was observed in both patterns of the larval feeding, continuous and discontinuous, at the three tested concentrations of L. sativum seed powder; while it was minimum (ca., 2.3 spermatophores) at the continuous larval feeding pattern with conc.1.00% (Table 2).As shown in Table 2, the mean spermatophore counts were remarkably increased in the experimental group when compared to that of the control group; such an increase seems to be neither dose-dependent nor larval feeding pattern, continuous or discontinuous.
Also, the findings in Table 2 reveal that, in terms of both the average spermatophore counts and the average egg counts, B. mori female moths that mated once (averagely1.37-time)lay significantly fewer eggs (mean = 450.63eggs; in the control group) than the females mated averagely from 2 to 3 times (mean = 731.16-1364.79eggs; in the experimental group).Thus, the increase in spermatophore numbers increases the average egg yield or fecundity of B. mori female moth by up to 2-3 times (62.26-202.87%)when compared to that of the control group.Therefore, it seems that B. mori female fecundity is dependent upon mating frequency.

Reproductive index
In general, L. sativum seed powder yielded large pupae and highly fecund moths (i.e., females or males) that laid profuse and large-sized eggs or mated more than twice compared to that of the controls (Table 2).B. mori adult females obtained from the untreated mulberry leaves-fed larvae (the controls) had the highest reproductive index (0.00180), revealing less efficiency in utilizing larval nutritive resources for reproduction (i.e., low mean values for egg count and size; Tables 2, 5).The reproductive index was estimated by dividing the mean value of the pupal weight (gm) by the corresponding value of the fecundity (no. of eggs laid per female).The enrichment of mulberry leaves with L. sativum seed powder led to lower values for the reproductive indices, ranged from 0.00080 to 0.00165, indicating better efficiency in utilization of larval resources for reproduction (Tables 2,  5).In terms of the reproductive index, it seems that B. mori female moths which having low indices, as in the experimental group, lay significantly large eggs; while those having a higher index, as in the control group, lay significantly small ones (Tables 3, 5).

Gain that could be benefited from the key physiological resources, protein-, lipid-, and carbohydrate-content, in B. mori female moths at death
Based on the present findings, it was fruitful to shed some light upon the fate of B. mori female moths after achieving their job in sericulture field, viz., egg production performance.In other words, what is the added value of B. mori female moths after their death in order not to be an addable burden from the environmental pollution point of view; plus loss of important nutrients such as, proteins, lipids, and carbohydrates.According to the literature review, all researches which are related to this subject have been carried out on determination of the abovementioned reserves, as valuable nutritional resources, in B. mori pupae.However, less attention (i.e., no study) has been paid to the total protein-, lipid-, or carbohydrate-content in B. mori female moths after their death.Therefore, the purpose of this experiment was to quantify the total protein-, lipid-and carbohydrate-content in the internal reproductive tract of B. mori female moths at death; hopefully to add value(s) besides their main performance, the egg production (e.g., providing certain manufactures with natural raw materials, e.g., pet feed formulations, from the moth abdomens, where the internal reproductive organs occupy most of the abdomen).Table 6 summarizes the relationship between the total protein-, lipid-, and carbohydrate-content, in the internal reproductive tract of B. mori female moths at death and egg production (fecundity) at indicated treatments.

The total protein content
The average values (± S.E.) of the total protein content (mg/gm) at the indicated treatments recorded a marginally significant decrease of about 0.80-4.80%over the control, except for the corresponding value (61.14 ± 0.13 mg/ gm) at both the highest conc.used (1.00%) and the continuous larval feeding pattern, where a marginal and statistically insignificant decrease (0.78%) over the control mean value (61.62 ± 0.06 mg/gm) was recorded.In the experimental group, the quantified mean values of the total protein content in the internal reproductive tract of B. mori female moths at death ranged from 58.69 to 61.14 mg/gm.As might be expected for a protein content to be decreased after the egg production performance; however, the relatively marginal reduction in the average protein content, in the experimental group, was greatly associated with remarkable average numbers of eggs laid Vol:.( 1234567890 6).Hence, it seems logical that the presence of L. sativum seed powder, in B. mori larval diet, would nutritionally have a strong physiological effect on both egg yield and egg size, towards yielding optimal egg counts and egg size (see Tables 3, 5, 6).Also, the decrease in the total protein content in the internal reproductive tract of B. mori female moths appeared to be a dose-dependent, and apt to be the least towards higher concentrations (i.e., > 0.25%).There was no statistically significant difference in protein content mean values at continuous and discontinuous larval feeding patterns (Table 6).

The total lipid content
The total lipid content in the internal reproductive tract of B. mori female moths, at death, was significantly higher in the experimental group than those in the control group by 2.05-17.13%,except for the corresponding value (17.76 ± 0.15 mg/gm) at both the highest conc.used (1.00%) and the continuous larval feeding pattern; where a significant decrease (22.51%) over the control mean value (± S.E.) (22.92 ± 0.23 mg/gm) was observed (Table 6).Also, data in Table 6 show that, in general, the mean value of the total lipid content was increased in a dosedependent way.In general, the quantified significant increase of lipid content, that ranged from 17.76 ± 0.15 mg/ gm to 26.85 ± 0.10 mg/gm, must ultimately be resulted from a well-enriched larval diet, since the B. mori female moths do not feed.That seems logical where B. mori female moths obtained from larvae fed on more nutritious mulberry leaves (i.e., enriched with L. sativum seed powder) carried over, in general, more lipid reserves from the larval stage than those obtained from larvae reared on a less nutritious diet (i.e., the control group).Moreover, as shown in Table 6, B. mori larvae reared on a nutrient-rich diet (the experimental group) gave rise to female moths which laid remarkably more eggs than those emerged from larvae reared on a less nutritious one (the control group).

The total carbohydrate content
The highest total carbohydrate content (6.34 ± 0.02 mg/gm) in the internal reproductive tract of B. mori female moths at death was detected at the continuous larval feeding pattern with L. sativum seed powder of conc.1.00%.This increase, by ca.756%, was significant as compared to the corresponding mean value (± S.E.) in the control group (0.74 ± 0.17 mg/gm); followed by an increase of ca.105% (1.52 ± 0.13 mg/gm) at the discontinuous larval feeding pattern with conc.0.25%, then by a non-significant increase of ca.14% at the continuous larval feeding pattern (0.85 ± 0.22 mg/gm) when compared to the control mean value in Table 6.Also, a significant or nonsignificant decrease, by ca.86, 87, and 22%, in the total carbohydrate content, was observed among the applied L. sativum concentrations, regardless of whether their larvae were fed continuously or discontinuously on mulberry leaves-enriched with L. sativum seed powder.At the larval continuous feeding pattern, the quantified increase in the total carbohydrate content was in a dose-dependent way (0.11 ± 0.04, 0.85 ± 0.22, and then 6.34 ± 0.02 mg/ gm); while at the discontinuous feeding pattern, there was no obvious trend for the increase or the decrease in the total carbohydrate content (Table 6).In general, the quantified mean values of the total protein, lipid, and carbohydrate contents were, to a large extent, statistically equal to, and higher or somewhat marginally lower than their corresponding mean values in the control group (Table 6).
The average body weights (± S.E.) of B. mori female moths, at death, resulting from L. sativum continuously fed larvae ranged between 0.30 ± 0.02 gm and 0.41 ± 0.03 gm; while those resulting from the larval discontinuous feeding pattern ranged from 0.28 ± 0.01 to 0.30 ± 0.02 gm, as compared to the corresponding mean value (± S.E.) of 0.34 ± 0.02 gm in the control group (Table 3).There were no significant differences (α level = 0.01) in female moth average body weights, at death, among both the experimental and the control groups.With this parameter, the observed insignificant increase (18.08%) or decrease (3.69-20.08%) in the experimental groups, over their control value, appears to be a dose-dependent.
In general, apart from L. sativum seed powder concentration used in the present study, the findings in Table 7 indicate that enrichment of mulberry leaves with L. sativum seed powder significantly increased all mean values of the subject economic parameters of B. mori, except for values of both the cocoon shell ratio percentage and the protein content in the female moth internal reproductive tract at death.In the experimental group, the mean values of the former parameter were significantly lower by nearly 18-21% than that of the control group; whereas a decrease, by up to 2.84%, in the mean values of the latter parameter was a marginally significant when compared to the control group (Table 7).On the one hand, there was no significant difference between the two experimental groups, viz., the continuous or the discontinuous larval feeding pattern, in the mean values of the following nine parameters (Table 7): cocoon shell weight, male or female longevity, spermatophore count per female, total protein content in the female moth internal reproductive tract at death, female moth body weight at death, and male body size (measured as forewing length, hind femur length or hind tibia length).On the other hand, there was a significant difference between both of the experimental larval feeding patterns, the continuous and the discontinuous, in mean values of the following eleven parameters (Table 7): five of them were in favour of the former feeding pattern (pupal weight, cocoon weight, egg surface area, egg volume, and total carbohydrate content in the female moth internal reproductive tract at death) and six parameters were in favour of the latter one (cocoon shell ratio%, female fecundity, total lipid content in the female moth internal reproductive tract at death, and female body size as measured by forewing, hind femur or hind tibia lengths).

Discussion
Many early and recent studies have been focused on the idea of how to nutritionally enrich mulberry leaves, Morus spp., with various botanical or animal supplements (e.g., botanical extracts, natural products, organic and inorganic compounds) in order to gain a better yield of B. mori silk production and/or egg production 14,15  www.nature.com/scientificreports/Empirically, B. mori physiological performance and its consequent outputs, in a form of a good yield of silk or eggs, is greatly influenced by nutrients in its larval diet, where the adult females do not feed 16,17 .
Based on personal observations on L. sativum seed powder nutritional value and its phagostimulant, as well as its antimicrobial properties, it was fruitful to nominate such nutritious seeds, as a fine powder, for enriching B. mori larval diet, mulberry leaves; hopefully to provide B. mori larvae with a more nutritious and economically cheap rearing diet.Chemically, L. sativum seed powder is a highly nutritive natural source of proteins, carbohydrates, lipids, vitamins, minerals, mucilage, fibers, steroids, antioxidants, sterols (β-sitosterol), and other essential nutrients or phytochemicals which enable it to be used in the enrichment of several foods and beverages or juices 18,19 .In the literature, there are no available reports on the effect of L. sativum seed powder on B. mori economic parameters; hence, the present study seems to be the first report in this concern.
As expected, the present findings had shed some light upon the promising role of L. sativum seed powder in sericulture.Firstly, obtaining highly fecund B. mori female and male moths.Secondly, regardless of the concentration used of L. sativum seed powder or the larval feeding pattern, viz., continuous or discontinuous, the female or the male moth reproductive performance was remarkable based on the percent increase over the control in mean values of the following parameters: fecundity (ca., 113-179%; or ca., a twofold to threefold increase), egg surface area (ca., 23-26%) or egg volume (ca., 44-49%), female or male longevity (ca., 8% or 29-30%, respectively), spermatophore counts per female (ca., 100-104%), and female or male moth body size, measured as forewing, hind femur or hind tibia lengths (ca., 5-25% for female moths or 5-10% for male moths).By comparing the effect of L. sativum-enriched larval food on B. mori egg yield (mean ± S.E.) per female moth in the experimental group (i.e., ranged between 958.41 ± 33.03 and 1255.92 ± 34.19 eggs, and 450.63 ± 17.38 eggs in the control group; see Table 7) and the corresponding mean values which have been reported in the available literature, the present L. sativum diet exhibited the best findings.In the literature, B. mori larvae which have been fed on mulberry leaves with or without nutritive supplements, the fecundity of their emerged female moths ranged between 329 and 835 eggs for treated leaves or between 220 and 750 eggs for the untreated ones 20,21 .
The chemical composition of L. sativum seed powder seems to be the main reason for the present good performance of B. mori in terms of cocoon economic parameters and moth reproductive success or fitness parameters.Therefore, a nutritional physiological explanation for the present unusually positive effects of L. sativum seed powder on the two vital physiological events, viz., the silk production and the egg production, might be in the following line: As reported by several authors, such important physiological events in B. mori life cycle depend on a number of influential factors; nutrition is one of these factors, where it plays an effectual role in sericulture 17,21 .
The percent increase over the control in B. mori pupal and adult economic parameters are evidently due to the improvement in the efficiency of B. mori larval diet by adding nutritious supplements to mulberry leaves.Although mulberry leaves per se are a complete diet for B. mori larvae; however, it is possible to improve the nutritional value of these leaves by enriching them with various inexpensive nutrients in order to enhance B. mori larval, pupal, and adult economic parameters.As might be expected, B. mori larvae would nutritionally benefit from such a handy enriched food viz., mulberry leaves per se and L. sativum seed powder.Hence, the phenomenal increase in the egg yield per B. mori female moth could be attributed to the enrichment of mulberry leaves with L. sativum seed powder where B. mori larvae have fed on a more nutritious food that would be rich in proteins, lipids, carbohydrates, vitamins, minerals, antioxidants, sterols, phospholipids, phytochemicals, etc.Thus, B. mori female moths obtained from well-nourished larvae laid more eggs (experimental group) than those from, relatively, poorly fed ones (control group).On the one hand, it is known that in the majority of insects, a proteinrich nutrient is the most vital factor affecting the total egg output 20 .On the other hand, protein is important for egg yolk production; where during the early stages of egg development, much protein is ingested and the intake of protein stimulates the corpora allata which secrete a factor leading to increased carbohydrate intake during the period of yolk deposition in the egg 22 .Therefore, these information may explain the remarkable increase in both the egg size and number laid per B. mori female moth in the presence of L. sativum seed powder in the larval food; as well as the unexpected reasonable increase in the total protein, lipid, or carbohydrate content in the internal reproductive tract of B. mori female moths at death; taking into consideration that B. mori moths do not feed, and the physiological or energetic costs concomitant with production of large eggs and being highly fecund moths rely heavily on their protein, lipid, and carbohydrate reserves that the female moths acquire through their larval feeding 20,23 .Expectedly, B. mori egg size can increase if physiologically important nutrients are at hand.In fact, this is the case in the present study where the enrichment of mulberry leaves with L. sativum seed powder seems to be the main cause beyond such desirable expectations, which could be supported by a similar opinion of Wiklund and Karlsson 24 in this concern.Jones 25 noted that larger females of the cabbage butterfly, Pieris rapae L. were more fecund than smaller ones, as well as the latter females tended to produce smaller eggs; hence, the egg size is dependent on female body size 26,27 .Consequently, the efficiency of L. sativum seed powder in B. mori larval diet has been translated into the following advantages, from a sericulture point of view, for both B. mori male and female moths: large body size, highly fecund and large-egged females, and a noticeable reproductive performance or fitness of male moth, in terms of significant increases in spermatophore counts per female moth.Similarly, Fox and Czesak 28 and Malik and Reddy 29 reported that better nutrition during larval period can greatly influence the female reproductive investment in both egg size and egg numbers.Thus, the larval diet and the female body size each, to a large extent, seems to have a physiological effect on both egg number and egg size 30,31 .Moreover, the availability of growth promoting compounds in botanicals (e.g., L. sativum seeds) may improve the bioavailability of nutrients for digestibility and may provide more energy for reproduction, leading to an increase in egg output of B. mori 32,33 .Inclusively, the larval feeding diet should be more important than the body size in determining the egg size 34,35 .On the other hand, in general, the diet responsible for production of greater numbers of eggs also produces large eggs 36 .Meanwhile, Honěk 26 stated that female body size is a principal constraint on insect potential fecundity.Further, it is commonly assumed that fecundity increases with body size in most animals and with insects this has been shown to be true 35,37  www.nature.com/scientificreports/Evidently, L. sativum seed powder-enriched mulberry leaves confer advantages not only to B. mori female moths but also to male moths in terms of an increase, over the control, in body size of ca.5-10%.Also, the present data reveal an increase, over the control, in spermatophore counts per female moth of ca.100-104%.Spermatophore count in B. mori dissected female moths seems to be useful in estimating the number of matings 38 per female moth.Findings of this study show that B. mori-female moth fecundity increases significantly, in the experiment group, with the number of spermatophores received (i.e., on average, ca.three spermatophores per female moth, as compared to nearly one spermatophore in the control moths).Papers of Eady et al. 39 and Arnqvist and Nilsson 40 could support the present results as they demonstrated that in many insects, mating frequency enhances female fecundity, as well as female that copulating repeatedly with the same male lay more eggs.The latter observation is congruent to the present case.Thus, both mating frequency and large body size are seemed to be important for B. mori male or female reproductive success.These observations are in agreement with Honěk 26 and Teng and Zhang 41 who also reported similar findings in their experimental lepidopterous species.From the literature, it has been shown that an insect body size is not the only parameter influencing male acceptance by female during courtship; meanwhile, a large body size should enhance male reproductive fitness or performance 42 .Also, the production of sex pheromone(s) released by male moths, that may facilitate female acceptance during courtship, oftenly requires precursors, e.g., alkaloids 43 .These precursors are present in plants 44 and insect male moths obtained these alkaloids through larval feeding 45,46 .Since L. sativum seed powder mainly contains alkaloids 47,48 therefore, the findings reported here may boost up the positive influential role of L. sativum seed powder in B. mori larval food.It seems that the male larval nutrition positively affects the reproductive fitness of both sexes of B. mori; taking into consideration the findings of Eisner and Meinwald 49 and LaMunyon and Eisner 50 that larger insect males provide greater amounts of alkaloids and nutrients that might both be good indicators of male and female fitness or reproductive success.However, a further study is certainly required to confirm the exact role of L. sativum-seed alkaloids as precursors of a male sex pheromone(s).
In addition to the observed remarkable increases in reproductive success or fitness parameters of B. mori male or female moths associated with their larval feeding on L. sativum seeds powder-enriched mulberry leaves, another bright side of this botanical additive relates to B. mori pupal economic parameters has been observed as well.The silk productivity, as measured by sericulture economic parameters like cocoon weight, cocoon shell weight, and pupal weight, showed significant increases in their mean values over the controls.Irrespective of L. sativum seed powder concentration used or B. mori larval feeding pattern, viz., continuous or discontinuous, the cocoon weight, the cocoon shell weight, and the pupal weight had increased significantly by a percent of nearly 30-38%, 7-10%, and 39-50%, respectively.However, the cocoon shell ratio (%) was higher by about 18-21% in the control group than in the experimental group.The latter result was expected and could be referred to the above-mentioned significant increase in pupal weights observed among L. sativum-fed group as compared to the corresponding significantly low weights in the control group.This finding is in agreement with those of Trivedy et al. 51 and Saad et al. 52 but with other nutritious additives.The present improvement in B. mori cocoon economic parameters could be attributed to the enrichment of mulberry leaves with extra nutrients which are amply available in L. sativum seed powder.Results of Gokavi et al. 53 and Al-Snafi 48 could boost the present findings as follows: these authors mentioned that the most abundant amino acid in L. sativum seeds is glutamic acid (ca., 19-24%) followed by aspartic acid (ca.,10-12%) then glycine, alanine, and serine acids (each ca., 4-6%).On the other hand, B. mori-larval feeding on mulberry leaves with high amounts of proteins and amino acids leads to turnover of proteins and amino acids which are concomitant with a high increase in glutamic and aspartic acids.Such amino acids are necessarily required for silk synthesis; not to mention that silk proteins of the cocoon shell (i.e., fibroin and sericin) are made up of polypeptide chain of amino acids, specifically alanine, glycine, and sericin.These amino acids are probably assimilated by B. mori larvae when they feed on protein-rich food 54,55 .Also, according to Murugan et al. 56 , higher levels of protein and the amino acid serine in B. mori silk glands are observed after the larval feeding on mulberry leaves enriched with botanical extracts (e.g., L. sativum seed powder) which are rich in proteins, amine acids, carbohydrates, minerals, vitamins, phytochemicals etc. Knowing that the fifth-larval-instar of B. mori utilizes about 65% of the absorbed nitrogen, therefore, nitrogenous compounds present in the larval feeding diet can have a great effect on larval growth and cocoon production 57,58 .Meanwhile, Lu and Jiang 59 have stated that B. mori larvae utilize 72-86% of amino acids from mulberry leaves, and more than a 60% of the absorbed amount is utilized for silk production.The authors added that absorption of amino acids in midgut of B. mori larvae depends on potassium ion.Additionally, the enrichment of mulberry leaves with the non-essential amino acids, aspartic (1 and 2%), alanine (0.5%), glycine (0.5%) or glutamic (1%) leads to increases in larval weight (by about 10-14%), silkworm economic parameters, larval food consumption, and absorption percentage 58,60 .Hence, amino acids are greatly important for the silk filament synthesis and could also improve the silk yield if added in adequate quantities 61 .L. sativum seeds are also a good source of vitamins, minerals, and antioxidants (due to their high content of phenolic compounds) 19,48 .Moreover, L. sativum seeds are rich in vitamins, C (ascorbic acid), A (β-carotene, provitamin A), E (tocopherols), and B vitamins such as thiamine (B 1 ), riboflavin (B 2 ), and nicotinic acid (niacin, B 3 ) 18,62 .Many reports have also stated that magnesium, calcium, phosphorus, potassium, iron, manganese, and zinc (amply present in L. sativum seeds 62,63 ) are essential elements required by B.mori to stimulate the metabolic activity 64 ; to improve the quality of the silk thread and cocoon parameters as well 65 .On the other hand, B. mori larvae which fed on a diet containing more minerals, divert minimum energy for maintenance; hence, the larvae can channel maximum energy for silk production 17,66 .
Vitamins and minerals, as other nutritive additives to mulberry leaves, play an important role in B. mori larval nutrition and hence increase the sericultural yield 61,67 .Since B. mori-fifth-larval-instar is the most important instar of silk production 68 , therefore, Ito 69 and Horie 70 mentioned that many members of vitamin B-complex, such as thiamine (B1), riboflavin (B2), and niacin (B3) have a direct or an indirect influential role on some economic parameters of B. mori as follows: Thiamine is important for metabolism and significantly increases cocoon weight, cocoon shell weight, and female moth fecundity; riboflavin improves the silk production, and www.nature.com/scientificreports/niacin is important for releasing energy from carbohydrates and lipids; it is also important in protein metabolism, and production of some hormones.Furthermore, vitamin C has been reported to enhance remarkably B. mori silk yield 67,71 .Besides, it acts as a phagostimulant or a gustatory stimulant 69,72 .The natural antioxidants like tocopherols (vitamin E), phytosterols, and carotenoids, which are available in L. sativum seeds 19,73 , have some growth promoting effects on B. mori larvae which result in increased silk output as reflected by improvement in cocoon parameters, i.e., cocoon weight, cocoon shell weight, and pupal weight 69 ; also have phagostimulant properties such as sterols 74 and their principal role in B. mori larvae seems to be nutritional [75][76][77] .Further, there is a possibility of conversion of sterols, in B. mori larval food, into active hormonal substances by B. mori larvae 77 .Also, B. mori-larval feeding diet's efficiency is highly increased in the presence of phospholipids 75,77 .L. sativum seeds are an excellent source of phospholipids (5.8gm per 100gm seeds 53 ).In summary, apart from the cocoon shell ratio (%), the present improvements in sericulture different parameters observed in the experimental group against the control group might be due to the nutritionally and physiologically important phytochemicals present in L. sativum seed powder, which proved to be essential for B. mori in addition to their natural food, mulberry leaves.The review of literature has revealed that these phytochemicals could act as a physiological stimulator of protein synthesis by tuning or stimulating the neuro-endocrine system in B. mori larvae and ultimately enhance the protein synthesis 56,78 which promotes both the silk synthesis, as evidenced the significant increases in the cocoon weight, the cocoon shell weight, and the pupal weight 17,55 , and the egg yield of the silkmoth 20,22 .Also, these phytochemicals could act efficiently as follows: (1) Phagostimulant (i.e., may increase the larval appetite leading to an increase in B. mori silk and egg productivity 7,77 ; (2) Antioxidant (i.e., may increase the absorption of the mutrients in the larval midgut leading to a high larval growth and consequently an increase in B. mori economic parameters 72,79 ; (3) Promoter of larval growth and silk production (i.e., due to their physiological stimulation 80,81 ; (4) Antimicrobial agent (i.e., for achieving a better rearing and productivity by avoiding B. mori-diseases which badly affect their health and productivity 6,82 .There is only one published paper in the literature which denoted with the positive effect of L. sativum seed oil on B. mori, where this oil had increased B. mori productive characters and can be utilized as an additive to increase the silk yield 83 .Based on all the above-mentioned findings, the present study or the corresponding ones in the literature, the phenomenal egg yield and the improvement in the cocoon economic parameters logically might be due to the nutritionally or physiologically valuable biochemical components amply available in L. sativam seed powder.These results are in agreement with the findings of many authors but with botanicals other than the garden cress, L. sativum seed powder 81,84 . In conclusion, the authors believe that the present study is the first to evaluate the efficacy of L. sativum seed powder as a promising food additive for improving the nutritional value of mulberry leaves and consequently enhancing the economic parameters of B. mori; hopefully to gain ground in the sericulture industry, hence increasing the national revenue.The ANOVA indicates that apart from B. mori shell ratio (%) in the experimental group, B. mori larval feeding on L. sativum-enriched mulberry leaves significantly increased all mean values of the different economic parameters evaluated in the experimental group comparing with their corresponding values in the control group.In general, the present study indicates that B. mori moths, which do not feed, are efficiently able to utilize the stored physiological resources or reserves (i.e., protein, lipid, and carbohydrate) gathered during the larval stage and remarkably enhanced the reproductive output or performance of their male or female moths.The latter parameter is translated into phenomenal high egg counts, high spermatophore counts, large eggs, large body size, and, in general, a reasonable content of protein, lipid, and carbohydrate in the internal reproductive tract of B. mori female moths at death.Hence, as being a good natural source of protein, lipid, and carbohydrate, B. mori female moth bodies at death could be nominated as a raw material in certain manufactures like pet feed formulations instead of discarding them as a source of environmental pollution.On the other hand, the enrichment efficacy of mulberry leaves with L. sativum seed powder is also translated into significant increases in B. mori cocoon economic parameters such as cocoon weight, pupal weight, and cocoon shell weight.Consequently, in sericulture field, great expectations of L. sativum seed powder seem to be promising. https://doi.org/10.1038/s41598-024-67128-0

Table 1 .
Effect of continuous (c) or discontinuous (d) feeding of B. mori larvae on mulberry leaves enriched with the indicated concentrations of L. sativum seed powder on certain economic parameters of their cocoons*.Values followed by different letters on the same row are significantly different at α level = 0.05; ANOVA followed by Duncan's multiple range test.*Sample size (n) = 129.**Positive or negative values in parentheses represent the percent increase or decrease, respectively, over the control.***7-days post cocoon spinning.

Table 4 .
Summary of the outcome of the present treatments addressing the effect of enrichment of mulberry leaves with L. sativum seed powder on certain B. mori-pupal economic parameters.*% change (increase) over control = treatment − control/control × 100.**A marginally non-significant decrease over the control.

Table 5 .
Summary of the outcome of the present treatments addressing the effect of enrichment of mulberry leaves with L. sativum seed powder on certain B. mori-moth fitness parameters.*% change (increase) over control = treatment − control/control × 100.B.

Table 6 .
The physiological cost(s) of being a highly fecund-B.mori female moths based on the quantified physiological resources, protein-, lipid-, and carbohydrate-content (mg/gm) in their internal reproductive tract at death.Values followed by different letters on the same row are significantly different at α level = 0.05; ANOVA followed by Duncan's multiple range test.*Measured immediately after female moth death.**Positive or negative values in parentheses represent the percent increase or decrease, respectively, over the control.

Table 7 .
Summary of the outcome of the present treatments of B. mori larval feeding, continuously (c) or discontinuously (d), on mulberry leaves enriched with the garden cress (L.sativum) seed powder (regardless of the concentration used) on some desired parameters in sericulture.*Values followed by different letters on the same row are significantly different at α level = 0.05; ANOVA followed by Duncan's multiple range test.**The total protein, lipid or carbohydrate content (mg/gm) in female moth internal reproductive tract at death.***Number of eggs laid per female.